Structural analysis of the phimotic prepuce in patients with failed topical treatment compared with untreated phimosis

ARTICLE INFO _______________________________________________________________________________ Vol. 38 (6): 802-808, November December, 2012 IBJU | STRUCTURAL ANALYSIS IN THE PHIMOTIC PREPUCE 803 Circumcision at childhood must be performed under general anesthesia. Moreover, the procedure itself is not free of risks, presenting complication rates up to 34% (3,4). The most common complications of circumcision are hemorrhage, urethral meatus and preputial ring stenosis, and even glans amputation (5). Besides these problems, circumcision presents considerable costs, which could approach a mean of US$ 1,920.00 per procedure, according to recent reports (6,7). The clinical treatment of phimosis with topical corticosteroids has been proposed as an alternative to surgery in the beginning of 1990, demonstrating acceptable results (8-10) and low cost (11). Since then, several authors have presented results varying from 67% to 95% of sucess, independently of the patients’ age. The most common used topical treatments were betamethasone, clobetasol, diclofenac sodium, mometasone furoate 0.05% and triamcinolone acetate (10,12,13). Recent randomized prospective studies have confi rmed that topical use of corticosteroids as treatment for phimosis is superior to placebo (14,15). Studies on the foreskin structure in patients with phimosis are scarce. Also, to our knowledge, there are no studies on histological alterations of the foreskin after topical corticosteroids treatment. Therefore, the objective of the present work was to evaluate the possible histological alterations in the foreskin of patients with phimosis submitted to topical application of an association of betamethasone 0.2% and hyaluronidase cream. MATERIALS AND METHODS The present work received institutional committee review and parental approval. This work was carried out in accordance with the ethical standards of the institutional committee responsible for human experimentation. We studied 60 patients ranging from 3 to 10 years (mean 4.5), during the period from January 2006 to October 2010. All patients presented true phimosis with foreskin stenosis and attended the pediatric urology ambulatory care to be submitted to circumcision. All 60 patients presented total impossibility of foreskin retraction, which characterizes the type A foreskin anatomy according to Marques (16). The exclusion criteria were patients with less than 3 years of age, patients without true phimosis and patients with clinical suspicion of balanitis xerotica obliterans. To the parents of the boys referred for circumcision we offered an alternative clinical management of the phimosis. The regimen proposed was a topical application of an association of betamethasone 0.2% and hyaluronidase (150 units of turbid retention) cream on the phimotic ring. The parents were oriented to perform gentle traction of the foreskin until the stenotic ring could be seen. The cream was applied twice a day, for 6 consecutive weeks, associated with appropriate hygiene of the penis. The children had a follow up every 3 weeks in our ambulatory. The parents of 7 children did not agree with the clinical treatment and opted for circumcision directly. These 7 patients served as the control group and the fi nal treated sample was composed of 53 patients. The topical treatment was considered successful if the patient was able to expose the glans completely. We considered failure if the glans could not be exposed after treatment or if occurred foreskin infection during treatment. In these cases, circumcision was performed. The patients submitted to topical treatment were followed up for 6 months. Histological Analysis In controls and in those cases on which the patients failed treatment and were submitted to circumcision, the foreskin was fi xed in a 10% buffered formalin solution, routinely processed for embedding in paraffi n and 5-μm thick sections were obtained. The sections were stained with Picro-Sirius Red, for qualitative analysis of the collagen under microscope of polarization and Weigert’s Resorcin Fuscin with previous oxidation by oxona, for characterization and quantifi cation of the elastic system fi bers. Quantitative Analysis From each specimen (foreskin), 5 different sections were randomly selected. From each section, 5 random fi elds were analyzed, totaling 25 fi elds (test areas) for each specimen. The data on IBJU | STRUCTURAL ANALYSIS IN THE PHIMOTIC PREPUCE 804 elastic system fi bers were expressed as volumetric density (Vv %). The sections were observed with X400 magnifi cation using an Olympus light microscope coupled to a video camera Sony CCD, and the images transferred to a Sony monitor KX14-CP1. The selected histological areas were then quantifi ed using M42 test-grid system on the digitized fi elds (17,18). (Figure-1). All numerical results are presented as mean ± standard deviation. The data were analyzed with the Graphpad software. To compare the quantitative data in both groups (controls and treated) and the outcomes, the Student’s t-test was used (p < 0.05 was considered signifi cant) (19).


INTRODUCTION
The foreskin is a specialized and innervated mucous-cutaneous tissue that covers and protects the glans.After 3 years of age, cysts of keratin are formed below the foreskin adherences and together with intermittent erections are able to enlarge the phimotic ring, exposing the glans.Around 80 to 90% of the boys that were not circu-mcised became capable to expose the glans after 3 years of age (1).
Pathological phimosis is characterized by a foreskin fi brotic ring with adherences that does not allow the exposition of the glans (1).This alteration hinders adequate penile hygiene that favors foreskin infections, repeated urinary infections, sexually transmitted diseases and, at adult age, penile carcinoma (2).

Structural analysis of the phimotic prepuce in patients with failed topical treatment compared with untreated phimosis ___ _______________________________________________ ____________________________________________
Circumcision at childhood must be performed under general anesthesia.Moreover, the procedure itself is not free of risks, presenting complication rates up to 34% (3,4).The most common complications of circumcision are hemorrhage, urethral meatus and preputial ring stenosis, and even glans amputation (5).Besides these problems, circumcision presents considerable costs, which could approach a mean of US$ 1,920.00 per procedure, according to recent reports (6,7).
The clinical treatment of phimosis with topical corticosteroids has been proposed as an alternative to surgery in the beginning of 1990, demonstrating acceptable results (8-10) and low cost (11).Since then, several authors have presented results varying from 67% to 95% of sucess, independently of the patients' age.The most common used topical treatments were betamethasone, clobetasol, diclofenac sodium, mometasone furoate 0.05% and triamcinolone acetate (10,12,13).Recent randomized prospective studies have confi rmed that topical use of corticosteroids as treatment for phimosis is superior to placebo (14,15).
Studies on the foreskin structure in patients with phimosis are scarce.Also, to our knowledge, there are no studies on histological alterations of the foreskin after topical corticosteroids treatment.Therefore, the objective of the present work was to evaluate the possible histological alterations in the foreskin of patients with phimosis submitted to topical application of an association of betamethasone 0.2% and hyaluronidase cream.

MATERIALS AND METHODS
The present work received institutional committee review and parental approval.This work was carried out in accordance with the ethical standards of the institutional committee responsible for human experimentation.
We studied 60 patients ranging from 3 to 10 years (mean 4.5), during the period from January 2006 to October 2010.All patients presented true phimosis with foreskin stenosis and attended the pediatric urology ambulatory care to be submitted to circumcision.All 60 patients presented total impossibility of foreskin retraction, which characterizes the type A foreskin anatomy accor-ding to Marques (16).The exclusion criteria were patients with less than 3 years of age, patients without true phimosis and patients with clinical suspicion of balanitis xerotica obliterans.
To the parents of the boys referred for circumcision we offered an alternative clinical management of the phimosis.The regimen proposed was a topical application of an association of betamethasone 0.2% and hyaluronidase (150 units of turbid retention) cream on the phimotic ring.The parents were oriented to perform gentle traction of the foreskin until the stenotic ring could be seen.The cream was applied twice a day, for 6 consecutive weeks, associated with appropriate hygiene of the penis.The children had a follow up every 3 weeks in our ambulatory.
The parents of 7 children did not agree with the clinical treatment and opted for circumcision directly.These 7 patients served as the control group and the fi nal treated sample was composed of 53 patients.
The topical treatment was considered successful if the patient was able to expose the glans completely.We considered failure if the glans could not be exposed after treatment or if occurred foreskin infection during treatment.In these cases, circumcision was performed.The patients submitted to topical treatment were followed up for 6 months.

Histological Analysis
In controls and in those cases on which the patients failed treatment and were submitted to circumcision, the foreskin was fi xed in a 10% buffered formalin solution, routinely processed for embedding in paraffi n and 5-µm thick sections were obtained.The sections were stained with Picro-Sirius Red, for qualitative analysis of the collagen under microscope of polarization and Weigert's Resorcin Fuscin with previous oxidation by oxona, for characterization and quantifi cation of the elastic system fi bers.

Quantitative Analysis
From each specimen (foreskin), 5 different sections were randomly selected.From each section, 5 random fi elds were analyzed, totaling 25 fi elds (test areas) for each specimen.The data on elastic system fi bers were expressed as volumetric density (Vv -%).The sections were observed with X400 magnifi cation using an Olympus light microscope coupled to a video camera Sony CCD, and the images transferred to a Sony monitor KX14-CP1.The selected histological areas were then quantifi ed using M42 test-grid system on the digitized fi elds (17,18).(Figure -1).All numerical results are presented as mean ± standard deviation.
The data were analyzed with the Graphpad software.To compare the quantitative data in both groups (controls and treated) and the outcomes, the Student's t-test was used (p < 0.05 was considered signifi cant) (19).

RESULTS
From the 53 patients submitted to topical treatment with the association of betamethasone and hyaluronidase cream, 8 (15%) presented failure (6 could not expose the glans and 2 remained with foreskin stenosis) and were referred to circumcision.The foreskin of these patients was submitted to histological analysis and composed our treated group.The others 45 patients presented signifi cant improvement of the phimosis, with total exposure of the glans without foreskin stenosis.After 6 months of follow-up, these patients considered as success did not present recurrence of the phimosis.
We analyzed the alterations on the elastic fi bers and collagen of the foreskin.Elastic system fi bers were analyzed in the foreskin of the control (n = 7) and treatment failed (n = 8) groups.Figure-2 shows the elastic system fi bers analysis in the foreskin.One may note in Figure -2B, in a patient who failed the topical treatment, an apparent lower amount of elastic fi bers when compared to controls (Figure -2A).The quantifi cation demonstrated that the mean volumetric density of elastic fi bers in the control group was 14.60% (11.06 to 21.64%) and in the group who failed treatment was 10.34% (3.45 to 17.9%); nevertheless, the difference was not signifi cant (p = 0.056).Data on the stereological quantifi cation of elastic system fi bers in the foreskin are presented in details on Table-1.
The Figure-3 evidences the alterations on the concentration of the collagen of the two groups studied, through the PicroSirius Red stain (with polarization).We can see the alteration on the pattern of the collagen of the patients submitted to the treatment with the cream.In Figure -3B,   one may observe the increase of the collagen type III (colored in green), on patients submitted to treatment with topical steroid.

DISCUSSION
The physiological phimosis affects 96% of the newborns and its incidence reduces with age.At 3 years of age, 10% of the patients present phimosis and at 14 years of age, this incidence affects only 1% (20).The natural process of enlargement of the prepuce can suffer alterations when facing episodes of balanoposthitis and lesion of the prepuce by traction of the ring that leads to the formation of a healing fi brosis and the impossibility to expose the glans.
The clinical treatment of the phimosis with topic corticosteroids is well accepted by the parents, since it is a simple procedure, presents low costs and risks, no side effects and a good compliance to the treatment when the guardians of the patients are well oriented (14,15).In our studies we did not have reports of signifi cant side effects and all fi fty-three patients submitted to clinical treatment completed the six weeks of treatment.
The success rate of the phimosis topic treatment with corticosteroids is signifi cant, with satisfactory results (67%-95%) (8)(9)(10)14,15).The success rate of the topical treatment of the phimosis is lower when smaller concentrations of betamethasone are used (21).We opted for the use of a larger concentration of betamethasone (0.2%) associated with hyluronidase.This larger concentration of corticosteroids does not present a larger rate of side effects and could enhance the success rate of the treatment.After six weeks of treatment, among the fi fty-three patients treated, 85% showed signifi cant improvement and were not submitted to surgery, demonstrating the effi ciency of the treatment.
Betamethasone is one of the substances that present better rates of effi ciency as a topical treatment of phimosis (20,22), therefore the chosen medication in this study.Corticosteroids reduce the arachidonic and hydroxyecosatetranoic acids on the reproductive infl ammatory skin disease, inhibiting the liberation of prostaglandins and enlarging the level of dismutase super-oxida-tive activity, being able to liberate anti-oxidants (20).Side effects, such as the suppression of the hypothalamo-pituitary-adrenal axis or cutaneous atrophy, may ocurr; nevertheless, the doses used in the topical treatment of the phimosis are not suffi cient to lead to this type of complications (1).
Hyaluronidase is an enzyme obtained from cattle testicles, which depolymerize hyaluronic acid, a mucopolysaccharide that is present in the interstitial tissue spaces (conjunctive tissue of the preputial adherences).The depolimerization of the hyaluronic acid reduces the inter-cellular viscosity, allowing the tissue to remain available to the dispersion of substances.Besides that, hyaluronidase presents elastic cohesion functions, being applied with the objective of cleaning the adherences and also because it presents the effects of a local anesthesia.
Collagen and elastic fi bers are the fi brotic components of the cellular matrix and are related to pathological alterations in different tissues.In our studies, we noticed a reduction of volumetric density of the elastic fi bers of the patient´s prepuces submitted to topical treatment, with corticosteroids + hyaluronidase.
The reduction of elastic fi bers of the prepuce in patients submitted to topical treatment with the cream was not signifi cant but is typical of the healing process of this treatment (23).The increasing concentration of elastic fi bers is related to a larger widening of the tissues.For an easy exposure of the glans, the prepuce needs a larger concentration of elastic fi bers (24).The reduction of the elastic fi bers in the prepuce of the patients presenting phimosis and submitted to a betamethasone + hyaluronidase treatment is similar to what occurs in the healing process and could be related to a larger diffi culty of the exposure of the glans; these patients did not report infections of the prepuce.
Topical treatment with steroids would intuitively reduce tissue scarring/fi brosis, and therefore is the mechanism by which the treatment would be successful.The interesting question to ask would be why did this cohort of patients have a signifi cant reduction in elastic fi bers but still fail topical treatment?These alterations of the elastic system could be involved on the therapeutic failu-re of the cream applied; nevertheless, to confi rm this hypothesis, it would be necessary to analyze the prepuces of the patients that received the treatment which obviously, for ethical reasons, is not possible.
The use of the betamethasone + hyaluronidase cream led to an evident alteration in the distribution of the collagen in the prepuces submitted to this topical treatment.There was a reduction of the collagen in the prepuce of the patients that made use of it.Nevertheless, when we analyzed the prepuces with the Picrosirus stain, it could be noticed a clear alteration in the distribution of the collagen, with and increase of collagen type III.The greenish fi bers that appear under the Picrosirus stain characterize the collagen type III, a recent collagen which is probably produced by muscular retraction.In the control group there was a predominance of a clear reddish color that characterizes the predominance of the collagen type I.It is very diffi cult to say, without studying the histology of the patients that made use of the cream and did not need a surgery, if the alterations of the collagen occurred due to induced alterations provoked by the cream or due to tissue alterations of the prepuce resulting from local infections.

CONCLUSIONS
The topical treatment of phimosis with betamethasone 0.2% + hyaluronidase was effective with a success rate of 85%.Patients in whom topical steroid treatment failed had fewer elastic fi bers, which characterize the healing processes, as well as an amplifi cation of the collagen type III, a recently found collagen that is associated with muscular retraction.The betamethasone + hyaluronidase cream leads to signifi cant histological alterations in the prepuce.

Figure 1 -Table 1 -
Figure 1 -Quantifi cation of elastic fi bers: The quantifi cation was obtained using the M42 Test system.Photomicrograph of the prepuce of an 8-year old patient of the control group (without treatment).Weigert stain reduced from X400.

Figure 2 -Figure 3 -
Figure 2 -Analysis of the elastic fi bers: A) Photomicrography of the prepuce of a 3-year old patient not treated with topical steroid.It is possible to observe that the elastic fi bers (purple) are distributed over the whole fi eld.Weigert stain reduced X400.B) Photomicrography of the prepuce of a 3-year old patient submitted to topical steroid treatment.It is possible to oberserve the elastic fi bers (purple).Weigert stain reduced X400.